PROJECT SUMMARY/ABSTRACT An effective HIV-1 vaccine will likely require the elicitation of humoral immunity in vaccine recipients. Vaccination studies have shown proof-of-concept that pre-existing antibodies may have the potential to prevent infection or slow disease progression upon HIV-1 or SIV exposure. Mother-to-child transmission (MTCT) is a natural setting in which the characteristics of protective pre-existing antibodies can be studied. HIV-positive mothers passively transfer HIV-specific IgG to their infants in utero, and these antibodies remain in infant circulation for 6 months or more, a key period of breastfeeding exposure to HIV-1. Less than half of infants born to HIV-positive mothers acquire HIV-1 through MTCT whereas the majority remain uninfected, even without antiretroviral therapy. This proposal addresses the hypothesis that pre-existing passively-transferred maternal antibodies present in infant circulation protect the infant from MTCT of HIV-1. Specifically, this proposal focuses on the hypothesis that antibodies from non-transmitting mothers target specific epitopes that confer their infants with protection from MTCT of HIV-1. It was recently reported that passively-transferred antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC; a mechanism of killing infected cells) was associated with reduced risk of infant mortality among infected infants and with a trend toward protection from MTCT of HIV-1. This study used a unique cohort of samples from the Nairobi Breastfeeding Clinical Trial (NBT) in Kenya, which was conducted from 1992-1998 before antiretroviral therapy was the standard of care. Because pre-existing passively-transferred antibodies have been shown to play a role in infant outcomes in this cohort, this cohort is ideal for in-depth investigation of the characteristics of beneficial passively-transferred antibodies. In the first part of this proposal, antibody binding to a panel of HIV-1 antigens will be measured, and epitopes of antibodies from non-transmitting and transmitting mother-infant pairs will be compared to determine whether targeting certain epitopes is associated with reduced risk of MTCT. Because ADCC was shown to be associated with improved infant outcome, the second part of this proposal will investigate the epitopes of these ADCC-mediating antibodies. A competition assay will be used to test whether ADCC-mediating antibodies from non-transmitting mother-infant pairs target different known ADCC epitopes compared to transmitting pairs. The last part of this proposal will finely map epitopes of monoclonal ADCC-mediating antibodies isolated from singly-sorted B cells from a non-transmitting mother who had high viral load, and thus was at high risk for transmitting HIV-1 to her infant. The goal of finely mapping epitopes of ADCC-mediating antibodies from a high risk non-transmitting mother is to characterize protective ADCC-mediating antibodies in detail. Overall, these studies will shed light on epitopes of antibodies capable of providing protection from HIV-1 infection, which will help inform for future vaccine design, passive vaccination strategies, antibody-based therapy.